Project Summary/Abstract Development, tissue renewal and long term survival of multi-cellular organisms is dependent upon the persistence of stem cells that are quiescent, but retain the capacity to re-enter the cell cycle to self-renew, or to produce progeny that can differentiate and re-populate the tissue. Deregulated release of these cells from the quiescent state, or preventing them from entering quiescence, results in uncontrolled proliferation and cancer. Conversely, loss of quiescent cells, or their failure to re-enter cell division, disrupts organ development and prevents tissue regeneration and repair. Understanding the quiescent state and how cells control the transitions in and out of this state is of fundamental importance. And yet, we know relatively little about it, due to a lack of tools for identifying and studying quiescent cells in their natural setting. We propose to identify genes that influence the entry, maintenance and recovery from the quiescent state in budding yeast cells. The quiescent state of budding yeast shares many important features with that of higher cells and the cell cycle is fundamentally conserved. As such, the strategies for arresting and maintaining this non-dividing quiescent state are likely to be shared. Entry into quiescence requires a stable but reversible arrest in G1. We are defining the mechanism of this G1 arrest and, as expected, we find striking parallels with the transition to quiescence in higher cells. Using flow cytometry, we have shown that wild type yeast grown to stationary phase differentiate into at least three cells types, only one of which bears the properties of quiescent cells. We can track, quantify and purify these quiescent cells. We have observed considerable variation in the yield and longevity of quiescent cells in lab and wild yeast strains. We are taking advantage of this natural variation and new genomic approaches to identify polymorphisms in essential and non-essential genes that influence the longevity of quiescent cells or regulate the entry into this state. It is our hope that discoveries made in budding yeast will offer testable models for the regulation of these important pathways in metazoan cells.